This research focuses on Cu/Zn superoxide dismutase (SOD1), an important anti-oxidant enzyme that scavenges superoxide and predominantly localizes to the cytosol of eukaryotes. The function of SOD1 has been somewhat enigmatic because superoxide is thought to largely arise from mitochondria, where a second SOD (Mn SOD2) already exists. Through studies in bakers yeast, we have been exploring the biology of Cu/Zn SOD1. Previously, we discovered the CCS metallochaperone that inserts the catalytic Cu co-factor into SOD1. CCS co-localizes with SOD1 primarily in the cytosol, but recently we found that both proteins also reside in the intermembrane space of the mitochondria where active SOD1 may directly combat respiratory sources of superoxide. By exploiting yeast and mammalian systems, the current Aims are designed to unravel the striking link between SOD1 and the mitochondria. AIM l: To identify the sources of oxidative damage relevant to Cu/Zn SOD1. The role of mitochondria in causing rapid aging and oxidative damage to yeast lacking SOD1 will be determined. Metabolic sources of superoxide will be identified through a genetic screen. AIM 2: To understand the physiology of mitochondrial Cu/Zn SOD1. The functions of cytosolic versus mitochondria SOD1 will be differentiated and the mechanism of mitochondrial import of SOD1 probed. We will also begin to address the possible implications for mitochondrial SOD1 in SOD1-linked cases of Amyotrophic Lateral Sclerosis. AIM 3: To define the CCS-independent pathway of copper delivery for SOD1. Mammalian SOD1 acquires a limited level of copper independent of CCS. We will now test whether this pathway occurs in mitochondria and will employ yeast genetics to identify factor(s) other than CCS that activate mammalian SOD1 with copper.